ES2198514T3 - PRIMERS AND PROBES FOR THE DETECTION OF HEPATITIS C. - Google Patents

Abstract

THIS INVENTION PROVIDES OLIGONUCLEOTIDE PROBES FOR DETECTING NUCLEIC ACIDS OF THE HEPATITIS C VIRUS OF THE US PROTOTYPE, JAPAN AND HCV-C VAPES, AS WELL AS OLIGONUCLEOTIDE INITIATORS TO AMPLIFY SUCH NUCLE ACIDS. THE INVENTION ALSO PROVIDES METHODS AND KITS TO AMPLIFY AND DETECT SUCH NUCLEIC ACIDS. METHODS OF AMPLIFICATION PREFERIBLY INCLUDE THE REVERSE TRANSCRIPTION OF THE VIRAL GENOMIC RNA TO CREATE CADN AND THE SUBSEQUENT AMPLIFICATION OF CADN THROUGH THE POLYMERASE CHAIN REACTION. BELOW OLIGONUCLEOTIDE PROBES CAN BE USED TO DETECT THE PRESENCE OF AMPLIFIED DNA BY HYBRIDIZATION.

Description

Primers and probes for the detection of hepatitis C.

The present invention provides primers and Enhanced probes for the detection of hepatitis C virus, a causative agent of non-A hepatitis, hepatitis no-B. Such reagents especially include oligonucleotide primers for both reverse transcription of the viral RNA genome as for subsequent amplification by the cDNA polymerase chain reaction as well produced. Sequence oligonucleotide probes are provided specific for the detection of the amplified sequences of the HCV nucleic acid. Primers and probes are useful in the diagnostic test for the detection of HCV infection. This diagnostic test has important applications in both the clinical field as in the epidemiological.

Hepatitis C virus (HCV) is one of the viruses among an unknown number of agents responsible for non-A, non-B hepatitis (NANBH). Prototypic HCV was identified from a cDNA clone of a blood NANBH virus, obtained from the plasma of an infected chimpanzee, as described by Choo et al., 1989, Science 244 : 359-362. The nucleotide sequences of the HCV genes of this prototype are described in European Patent Publication No. 318,216; 388,232 and 398,748. The nucleotide sequence of the HCV genome was disclosed and compared to related viruses by Choo et al., 1991, Proc. Natl. Acad. Sci. USA 88 : 2451-2455.

Since then the sequences of other strains have been disclosed. The HCV genome shows a high degree of heterogeneity in the nucleic acid sequences of the isolates from each other. Isolation of the cDNA from the HCV RNA genome was published by Kubo et al., 1989, Nuc. Acids Res. 17 : 10367-10372. The authors constructed a reverse transcription primer based on the sequence disclosed by Choo et al., 1989, see above. The HCV genome fragment isolated showed 79.8% homology with the prototype HCV sequence. Obtained by a similar protocol, sequences from three different regions were disclosed by Takeuchi et al., 1990, Gene 91 : 287-291. The homology of the sequences with the prototype sequence was found to be low, namely 73.5% for one of the regions.

Kato et al., 1990, Proc. Natl. Acad. Sci. USA 87 : 9524-9528, disclosed the HCV genomic sequence of a strain isolated from Japanese patients. The sequence showed 77.4% homology with the HCV genome prototype in the region compared. A sequence of the complete coding region of the HCV genome published in Takamizawa et al., 1991, J. Virol. 65 (3): 1105-1113 showed 77% homology with the prototype HCV strain.

In Japan, two main strains, designated by the name of K1 and K2, have been studied based on a genome region of 400 nucleotides. These strains have a sequence homology with the low HCV prototype sequence of 67% (see Enomoto et al., 1990, Biochem. Biophys. Common Res. 170 (3): 1021-1025). The K2 strain could also be subdivided into two groups, designated as Ka and Kb, with approximately 20% variation of nucleotides between the groups and approximately 5% variation of nucleotides within each group.

Similar homologies have been made known with the prototype HCV sequence, by Kato et al., 1990, J. Clin. Invest. 86 : 1764-1767. From 15 patients, cDNA fragments were amplified and sequenced. The amplified portion, 37 nucleotides corresponding to positions 3525-3561 of the prototype sequence, showed 68-78% homology with the prototype HCV sequence.

HCV genomic RNA can be detected in sera by creating the cDNA from genomic RNA, amplifying the cDNA by the chain reaction method of the polymerase, and then probe with oligonucleotides of the specific sequence Because of the heterogeneity of the sequences between the different strains of HCV, the primers probes they should probably be strain specific, unless a region can be found in which the sequence is preserved at Through the strains. One of these conserved regions is in the 5 'end of the HCV genome.

The non-coding sequence of the 5 'terminal of the HCV genome was first disclosed by Okamoto et al., 1990, Japan J. Exp. Med. 60 (3): 167-177 and in EP-A-461,863. The comparison between two strains suggested that the 5 'terminal non-coding sequence is conserved from one strain to another. The conserved nature of the non-coding sequence of the 5 'terminal of the HCV genome was also described by Han et al., 1991, Proc. Natl. Acad. Sci. USA 88 : 1711-1715. Partial sequences of a region of 341 nucleotides obtained from 11 different HCV isolates from individuals on five continents were compared. Seven sequences had complete homology with the prototype sequence, the remaining four showed between one and five base mismatches.

Okamoto et al., 1990, Japan J. Exp. Med. 60 (3): 215-222, described primers for various regions of the HCV genome, including the conserved 5 'non-coding region. The primers selected from conserved regions successfully amplified the nucleic acid of most of the strains tested; primers chosen from heterogeneous regions amplified the nucleic acid of a small set of strains. However, the efficiency of amplification with these primers was low. The reference describes a two-stage PCR amplification; The second amplification cycle was performed on the previously amplified target region, using a second set of primers housed within the region amplified by the first set of primers.

A PCR amplification of two amplification cycles using two sets of primers was also described by Garzón et al., 1990, Lancet 335 : 1419-1422. A region encoding a non-structural protein (NS5) was amplified. Due to the heterogeneity of the sequences, there are HCV sequences not recognized by these primers (see Garson et al., 1990, Lancet 336 : 878: 879). Consequently, primers in the conserved 5 'non-coding region were tested, but in order to obtain sufficient sensitivity, two-stage amplification using sets of housed primers was still necessary. Amplification of the 5 'terminal region using a two-stage amplification with hosted primers was also described by Kanai et al., 1990, Lancet 336 : 245.

The amplification using two cycles of amplification with hosted primers is not only ineffective but It also increases the likelihood of contamination. The problems of Pollution are already well known in the specialty; the opening of the reaction tube to change the primers and add reagents between the amplification steps, the maximum possible should be avoided. The pollution problem is further compounded by the need of changing the reaction conditions between the passage of Initial reverse transcription and subsequent amplification by PCR

There is also a need for primer oligonucleotides for amplification of the HVC sequences, that each one is chosen from a region preserved so that all or almost all strains are amplified and amplification methods are sufficiently effective so that amplification with a set of primers is enough. There is also a need for oligonucleotides of the probe for the detection of the amplified cDNA are chosen from a conserved region between the two conserved regions with the which hybridize the primers. It is necessary that the protocol of the reaction and reagents allow reverse transcription and PCR using the same reagents, thereby eliminating the need to open the reaction tube during the process of amplification.

In addition, ten percent of cases are non-reactive with the prototype capsid and cover antigens, see Chaudhary et al., 1991, J. Clinical Microbiology 29 : 2329-2330 and Hosein et al., 1991, PNAS 8 : 3647-3651. Thus, the development of PCR-based diagnoses and antigens encoded by new isolates will increase the dependence of diagnostic tests with serological basis. The present invention meets these needs by providing primers, probes and methods for the detection of HCV.

Specific primers and probes of oligonucleotides of the specific sequence provided, may be employed in a polymerase chain reaction for the homogeneous reverse transcription (RT-PCR) that allows amplification and detection of RNA genomic sequences viral. The effectiveness of amplification using primers and methods of the present invention eliminates the need for a second PCR amplification cycle with built-in primers, and the high sensitivity of the resulting assay provides reliable nucleic acid detection.

An aspect of the invention relates to primers of specific oligonucleotides. The invention provides compositions comprising an oligonucleotide primer for the amplification of the HCV nucleic acid, wherein said primer is suitable for amplification of a sub sequence of an acid nucleic from a strain or isotype of HCV selected from the group formed by the strains of Japan, US and C9. The primers they provided the function of both reverse transcription of viral genome of ATN as of the subsequent amplification of the cDNA produced using the polymerase chain reaction. The primers hybridize with the sequences of conserved regions of the HCV genome and therefore bind to a wide variety of strains The effectiveness of amplification using primers contributed is enough to eliminate the need for a second PCR amplification cycle with hosted primers.

Another aspect of the invention relates to probes capable of detecting the presence of HCV genomic nucleic acid regardless of the strain. The probes are complementary to the regions conserved through the strains. The essays of diagnosis using the probes of the present invention can detect a large number of strains of HCV without the commitment of the specificity Thus, the invention provides compositions that comprise an oligonucleotide probe for the detection of presence of hepatitis C virus nucleic acids, where the probe is suitable for the detection of nucleic acid from a HCV strain or a variant strain selected from the group formed by the prototype strains of Japan, US and C9.

A third aspect of the invention relates to the kits These kits have a wide variety of shapes and can contain one or more reverse transcription reagents, amplification, or detection, e.g. ex. primers, probes, polymerases, glycosylases, buffers and nucleosides triphosphates.

Another aspect of the invention relates to methods for the amplification and detection of HCV RNA.

European Patent No. 92114115.6 (EP-A-529493) provides a kit for the detection of specific nucleic acid for an isolate of C9 hepatitis virus, the kit comprising a compartment which contains a nucleic acid probe that binds substantially to a subsequent nucleic acid virus HCV-C9.

In addition, European Patent No. 92114115.6 provides compositions comprising an acid sequence viral nucleus substantially homologous to SEQ ID No. 28. A clone of cDNA, PHCV-C9, which contains a viral sequence of this nature, referred to herein as isolated C9, has been deposited in the American Type Culture Collection and has Deposit No. 75265.

European Patent No. 92114115.6 also provides oligonucleotide probes and primers for detection of a specific nucleic acid sequence for the C9 isolate and related variants. The probes of the invention comprise, of Preference, a sub-sequence selected from the following group:

SEQ ID NO. 33 5'-TTGCCGGAAAGACTGGGTCCTTTC-3 ' (nt174-197)

SEQ ID NO. 3. 4 5'-CAAAAGAAACACAAACCGCCGCCC-3 ' (nt374-397)

SEQ ID NO. 35 5'-CCAGCCCATCCCGAAAGATCGGCG-3 ' (nt527-550)

SEQ ID NO. 36 (MY160) 5'-TGTCCGGTCATTTGGGCG-3 ' (nt216-233)

European Patent No. 92114115.6 provides in addition oligonucleotide primers for amplification of specific nucleic acid sequences for a C9 isolate. The primers of the invention to specifically amplify the variant C9 and related isolates preferably comprise a sequence of nucleic acid selected from the following group of primers input line:

SEQ ID NO. 37 5'-AAACCCACTCTATGTCCGGTC-3 ' (nt204-224);

SEQ ID NO. 38 5'-GTACGCCGGAATTGCCGGAAA-3 ' (nt163-183); Y

SEQ ID No. 39 5'-CCTCAAAGAAAAACCAAAAGA-3 ' (nt360-380);

for use with one of the following primers from the starting line:

SEQ ID NO. 40 5'-TGGCGTCTCCCACGCGGCTGG-3 ' (nt509-529); Y

SEQ ID NO. 41 5'-CTTTCCCCAGGACCTGCCGGT-3 '(nt 555-575).

European Patent No. 92114115.6 provides also probes that substantially bind sequences of the isolate C9 and previously identified isolates of Hepatitis C.

Probes in this category include:

SEQ ID NO. 42 KY150 5'-CATAGTGGTCTGCGGAACCGGTGAGT-3 '.

The invention further provides probes for the HVC sequence detection other than C9, more specifically the invention provides a method for detecting the presence of hepatitis C genomic nucleic acid, where said hepatitis C genomic nucleic acid is selected from group consisting of prototype strains from Japan, U.S. and C9, the which procedure consists of: (a) amplification of a subsequence of the nucleic acid; (b) mixture of the nucleic acid amplified with an oligonucleotide probe specific for the sub sequence in conditions under which the probe binds to the sub sequence for form a stable duplex hybrid; and (c) hybrid detection formed between the sub sequence and the probe.

The method may further comprise, before the step (a) the amplification step of a sequence sequence virus specific The amplification is preferably performed by the polymerase chain reaction method. The primers and probes mentioned above are preferably used in The methods of the invention.

To help a better understanding of the invention, several expressions are defined below:

`` Amplification reaction mixture '' is refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These include: enzymes, aqueous buffers, salts, target nucleic acid and deoxynucleosides triphosphates. Depending on the context the mixture it may be either a complete mixture of amplification reaction or Well an incomplete mix.

`` Amplification reaction system '' refers to any means in vitro for the multiplication of the cups of a nucleic acid target sequence. These methods include, but are not limited to, polymerase chain reaction (PCR) amplification, DNA ligase, QB RNA replicase, and transcription based amplification systems. These involve multiple amplification reagents and are described in more detail below.

`` Reaction tube (s) of amplification '' refers to a container suitable for containing amplification reagents. Generally, the tube is constructed of inert components so that they do not inhibit or interfere with the amplification system used. When the system requires a thermal cycle or a repeated cycle of heat and cool, the tube must be able to withstand the process cyclic and, mainly, precisely fit the wells of the thermal cycler.

`` Amplification reagents '' refer to the various buffers, enzymes, primers, deoxynucleosides triphosphates (both conventional and unconventional), and primers employees to carry out the amplification procedure selected.

`` Amplification '', which refers mainly to an `` exponential '' increase in nucleic acid target, is used in this study to describe both an increase linear as exponential of the number of a target sequence selected from nucleic acid.

`` Binds substantially '' refers to to complementary hybridization between an oligonucleotide and a target sequence and encompasses minor mismatches that can be adapted by reducing the strictness of the means of hybridization to achieve the desired priming for the polymerases of the PCR or hybridization signal detection.

The phrase `` biologically pure '' refers to material that is substantially or essentially free of components that normally accompany it as is the case with your native state For example, antibodies purified by affinity or monoclonal antibodies exist in a state Biologically purified

`` Hybridization '' refers to the union of two single-stranded individual nucleic acids by the mating of the complementary phases.

`` Nucleic acid '' refers to a polymer deoxyribonucleotide or ribonucleotide, both mono and double-stranded, which otherwise is limited, would cover the known analogs of natural nucleotides that can act as similar to the nucleotides found in the nature.

`` Nucleotide polymerases '' refers to the enzymes capable of catalyzing the synthesis of DNA or RNA from nucleoside triphosphate precursors. In the reactions of amplification of this invention, polymerases are dependent of a mold and typically add nucleotides to the 3 'end of polymer that is forming. Most preferred is that the Polymerase is thermostable as described in U.S. Pat. nº 4,889,818 and 5,079,352.

The term `` oligonucleotide '' refers to a molecule comprising two or more deoxyribonucleotides or ribonucleotides, such as primers, probes, nucleic acid fragments to be detected, and nucleic acid controls. The exact size of an oligonucleotide depends on many factors and the ultimate function or use of the oligonucleotide. The oligonucleotides can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol 68 : 90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol 68 : 109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Lett. 22 : 1859-1862; and the solid support method of US Patent No. 4,458,066.

The term `` primer '' refers to a oligonucleotide, both natural and synthetic, capable of acting as starting point of DNA synthesis under conditions in which induces the synthesis of an extension product of the primer that is complementary to a nucleic acid chain, is that is, in the presence of four different nucleoside triphosphates and a polymerization agent (i.e. DNA polymerase or transcriptase reverse) in an appropriate buffer and at a suitable temperature. A primer is preferably an oligodeoxyribonucleotide single chain The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 25 nucleotides Short primer molecules require generally cooler temperatures to form hybrid complexes sufficiently stable with the mold. A primer does not need reflect the exact sequence of the mold but it must be Complementary enough to hybridize with a mold.

In the described versions of the invention, they provide primers and probes of specific sequences. The experts in the specialty will find it obvious that with these versions can be prepared primers and additional probes of a specific sequence p. ex. the addition of nucleotides in the 5'or 3 'ends, which nucleotides are complementary to the target sequence or are incomplete to the target sequence. As long as the primer compositions serve as a point of initiation for extension on target sequences, and whenever primers and probes comprise at least 14 nucleotides Consecutive contents contained in the exemplified versions, These compositions are part of the scope of the invention.

The term `` primer '' can refer to more than a primer, particularly in the case where there is some ambiguity in the information regarding one or both ends of the target region to be amplified. For example, if a region shows significant levels of polymorphism in a population mixtures of primers that amplify sequences can be prepared alternatives. A primer can be marked if desired, incorporating to it a mark detectable by spectroscopic means, Photochemical, biochemical, immunochemical or chemical. For example, useful marks are 32 P, fluorescent dyes, electron-dense reagents, enzymes (such as those used currently in the ELISA analysis), biotin, or haptens and proteins for which antisera or antibodies are available monoclonal A brand can also be used to `` capture '' the primer, so that immobilization is facilitated either from the primer or from the primer extension product, such as the amplified DNA, on a solid support.

`` Probe '' refers to an oligonucleotide that is unites through the pairing of the complementary bases, to a Subsequence of a target nucleic acid. For an expert in specialty it will be evident that the probes will typically be attached substantially to target sequences that lack a complete complementarity with the probe sequence, depending on the Stricticity of hybridization conditions. The probes are mark preferably directly as p. ex. with isotopes, or it mark indirectly as p. ex. with biotin to which you can join a streptavidin complex later. Analyzing the presence or absence of the probe, the presence or absence of the target.

`` Recombinant '' when referring to a probe nucleic acid, refers to an oligonucleotide that is free of native proteins and nucleic acid typically associated with probes isolated from cells, which naturally contain the probe sequence as a part of its native genome. The Recombinant probes include those obtained by means of amplification such as PCR and genetic cloning methods in where bacteria are transformed with the recombinant probe.

The term `` reverse transcriptase '' refers to an enzyme that catalyzes the polymerization of deoxyribonucleoside triphosphates to form the products of primer extension that are complementary to an acid mold rubonucleic The enzyme starts the synthesis at the 3 'end of the primer and continue towards the 5 'end of the mold until the synthesis ends. Examples of suitable polymerization agents that convert the target RNA sequence into a sequence Complementary copy of DNA (cDNA), are reverse transcriptase of the avian myeloblastosis virus and the DNA polymerase of the Thermus thermophilus, a thermostable DNA polymerase with reverse transcriptase activity.

The terms `` oligonucleotide of a sequence specific '' and `` SSO '' refer to oligonucleotides that have a sequence, called `` hybridization region '', exactly complementary to the sequence to be detected, typically characteristic sequences of a particular allele or variant, the what is `` strict sequence hybridization conditions specific '', will hybridize only this target sequence exactly complementary. Relaxing the strictness of the conditions of hybridization will allow the disappearance of the sequences; the degree of disappearance tolerated can be controlled by proper adjustment of the hybridization conditions. In depending on the sequences to be analyzed, one can be used or more oligonucleotides of the specific sequence. The terms `` probe '' and `` SSO probe '' are used by exchanging them with `` SSO. ''

A `` specific sequence of '' a viral isolate particular is a unique sequence of the isolate, which is, not shared by other previously characterized isolates. A probe containing a complementary sequence to a sequence specific of an isolate will typically not hybridize with the portion corresponding genome of other isolates under conditions strict (eg washing the solid support in 2xSSC, 0.1% SDS at 70 ° C).

A specific antigen or epitope of an isolate particular is one that is unique to the isolated and is not shared by other previously characterized isolates. An immunoglobulin generated against the antigen or epitope will not interreact with antigens in isolates previously characterized in assays standard, as p. ex. ELISA

The term `` substantially identical '' indicates that two or more nucleotide sequences have in common a large Most of its sequences. Generally, this will be at least approximately 90% of its sequence and preferably approximately 95% of its sequence. Another indication that sequences are substantially identical is if they hybridize with the same nucleotide sequence under strict conditions (see e.g. Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1985). Strict conditions are determined based on the sequence and they will be different in different circumstances. Generally the strict conditions are chosen so that they are approximately 5 ° C below the thermal melting point (Tm) of the sequence specific to a defined ionic strength and pH. The Tm is the temperature (at a certain ionic power and pH) at which the 50% of the hybrid target sequence with a perfectly probe partner. Typically, the strict conditions will be those in which salt concentration is at least approximately  0.2 molar at pH 7 and the temperature is at least approximately 60 ° C.

`` Subsequence '' refers to a sequence of nucleic acids comprising a part of a longer sequence of nucleic acids.

The term `` target region '' refers to the region of a nucleic acid that is being analyzed and that can include a polymorphic region.

The term `` thermostable enzyme polymerase '' is refers to an enzyme that is relatively heat stable and catalyzes the polymerization of nucleoside triphosphates to form primer extension products that are complementary to one of the nucleic acid chains of the target sequence. The enzyme begins synthesis at the 3'end of the primer and continues towards the 5 'end of the mold until the synthesis ends. A purified thermostable polymerase enzyme is described with more detail in U.S. Pat. No. 4,889,818.

Figure 1 represents an alignment of the C9 sequence of the new HCV variant, four related variants, and isolated HCV prototypes from Japan and U.S.

Hepatitis C virus is a small virus RNA that contains an individual RNA molecule, meaning positive, approximately 10,000 nucleotides in length. The genome contains a single, long open reading frame that is believes it translates into a single, large polyprotein that then It is processed. The open reading frame starts at the nucleotide 343 (using the numbering system from the virus prototype) followed by an untranslated region (UTR). Sequence 5'UTR is relatively preserved and may be important in the viral replication and regulation. The 5 'end of the region Modifier is also preserved. Certain primers and probes of the present invention hybridize in the conserved region at the end 5 'of the HCV genome.

The oligonucleotide sequences of the hybridization regions of the primers and probes of the invention They are presented later. Experts in the specialty will be given note that an oligonucleotide sequence used as a region of hybridization of a primer can also be used as a region of probe hybridization. The suitability of a primer sequence to be used as a probe depends on the characteristics of primer hybridization. Similarly, an oligonucleotide used as a probe it can be used as a primer.

The oligonucleotides represented in Table 1 they are primers or probes of positive sense (input line). The listing is in the order based on the position of the 3 'end of the oligonucleotide when hybridized with a genomic nucleic acid. The oligonucleotides that hybridize as close to the 5 'end of the Genome are the first on the list.

TABLE 1

         \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ + # \ hfil \ + \ hfil # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
  Oligo \ + Listing \ + Sequences \ +
 Position \ cr \ + Sequences \ + \ + (nt) \ cr KY65
\ + SEQ ID NO: 1 \ + 5'-CCAAGCTTCACCATAGATCACT \ +
16-29 \ cr KY79 \ + SEQ ID NO: 2 \ +
5'-GGCGACACTCCACCATAGATCACT \ +
6-29 \ cr KY98 \ + SEQ ID NO: 3 \ +
5'-CCAAGCTTAGATCACTCCCCTGTGAGGAACT \ +
21-44 \ cr KY96 \ + SEQ ID NO: 4 \ +
5'-CCAAGCTTCACGCAGAAAGCGTCTAGCCAT \ +
50-74 \ cr KY80 \ + SEQ ID NO: 5 \ +
5'-GCAGAAAGCGTCTAGCCATGGCGT \ +
56-79 \ cr KY144 \ + SEQ ID NO: 6 \ +
5'-ACGCAGAAAGCGTCTAGCCATGGCGT \ +
54-79 \ cr KY83 \ + SEQ ID NO: 7 \ +
5'-CCTCCAGGACCCCCCCTCCCGGGAGAGCCA \ +
99-128 \ cr KY84 \ + SEQ ID NO: 8 \ +
5'-GAGTACACCGGAATTGCCAGGACGACC \ +
149-175 \ cr KY85 \ + SEQ ID NO: 9 \ +
5'-ACCCGCTCAATGCCTGGAGAT \ +
194-214 \ cr KY67 \ + SEQ ID NO: 10 \ +
5'-CGAAGCTTGCTAGCCGAGTAGT \ +
236-250 \ cr KY80 \ + SEQ ID NO: 11 \ +
5'-CCGCAAGACTGCTAGCCGAGTAGT \ +
227-250 \ cr KY88 \ + SEQ ID NO: 12 \ +
5'-GTTGGGTCGCGAAAGGCCTTGTGGT \ +
251-275 \ cr KY86 \ + SEQ ID NO: 13 \ +
5'-GGTGCTTGCGAGTGCCCCGGGAGGTCTCGT \ +
288-317 \ cr KY87 \ + SEQ ID NO: 14 \ +
5'-GACTTCCGAGCGGTCGCAACCTCG \ +
482-505 \ cr}
      

Table 2 includes the oligonucleotides that they act as primers or as negative sense probes (line of exit). The same internal sort is used as in the table one.

TABLE 2

         \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ + # \ hfil \ + \ hfil # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
  Oligo \ + Listing \ + Sequences \ +
 Position \ cr \ + Sequences \ + \ + (nt) \ cr KY153
\ + SEQ ID NO: 15 \ + 5'-CCCAACACTACTCGGCTAGCAGTCT \ +
232-256 \ cr KY149 \ + SEQ ID NO: 16 \ +
5'-AAGGCCTTTCGCGACCCAACACTACT \ +
245-278 \ cr KY148 \ + SEQ ID NO: 17 \ +
5'-CACAAGGCCTTTCGCGACCCAACACT \ +
248-273 \ cr KY78 \ + SEQ ID NO: 18 \ +
5'-CTCGCAAGCACCCTATCAGGCAGT \ +
276-299 \ cr KY145 \ + SEQ ID NO: 19 \ +
5'-CACTCGCAAGCACCCTATCAGGCAGT \ +
276-301 \ cr KY95 \ + SEQ ID NO: 20 \ +
5'-GGGAATTCGCAAGCACCCTATCAGGCAGT \ +
276-298 \ cr KY100 \ + SEQ ID NO: 21 \ +
5'-CGAGGTTGCGACCGCTCGGAAGT \ +
483-505 \ cr KY110 \ + SEQ ID NO: 22 \ +
5'-AGGTTGCGACCGCTCGGAAGT \ +
483-503 \ cr KY109 \ + SEQ ID NO: 23 \ +
5'-AATGCCATAGAGGGGCCAAGG \ +
573-593 \ cr KY111 \ + SEQ ID NO: 24 \ +
5'-ATTGCCATAGAGGGGCCAAGG \ +
573-594 \ cr KY99 \ + SEQ ID NO: 25 \ +
5'-CAGAATTCATTGCCATAGAGGGGCCAAGGAT \ +
570-592 \ cr KY68 \ + SEQ ID NO: 26 \ +
5'-CAGAATTCGCCCTCATTGCCAT \ +
586-599 \ cr KY82 \ + SEQ ID NO: 27 \ +
5'-CCCACCCCAAGCCCTCATTGCCAT \ +
586-610 \ cr}
      

Several oligonucleotides possess regions of modified hybridization to include a restriction site towards the 5 'end of the molecule. The restriction site is entered in the amplified product when one of these oligonucleotides is used as a primer. The initial hybridization conditions are choose in such a way that the mismatches of the base pairs around the restriction site, they are tolerated. The mismatches near the 5 'end are better tolerated than close to the 3 'end of a primer. KY65 oligonucleotides (SEQ ID NO. 1), KY98 (SEQ ID NO. 3), KY96 (SEQ ID NO. 4), and KY67 (SEQ ID NO. 10), are starting line primers and contain a Hindi site. Oligonucleotides KY95 (SEQ ID NO. 20), KY99 (SEQ ID DO NOT. 26), and KY68 (SEQ ID NO. 27), are starting line primers and They contain an EcoRI site. The incorporation of such sites restriction on the amplified product facilitates the cloning of the amplified product

European Patent No. 92114115.6 provides also a new HCV isolate. The majority of isolates are related to one or two strains of HCV. The first is a strain U.S. prototype described in the European Patent Publication No. 318,216, 388,232, and 398,748. The second is a Japanese strain described by Kato et al., (see above). The two strains differ up to 30% in regions of putative non-structural genes, but show a homology greater than 98% in the 5 'region translated, and a homology of 92% in the 5 'portion of the gene of the capsid or core.

The isolate of European Patent No. 92114115.6, which is called C9, is more distantly related to both strains This new isolate is 93% similar to the region of 5 'consensus not translated. It has only one homology approximately 85% in the 5 'portion of the gene region of the nucleus with both the prototype or the Japanese strain (see table 3, later).

The isolated C9 (SEQ ID NO. 28) and related isotypes, R45 (SEQ ID NO. 30), R43 (SEQ. ID NO. 32), R110 (SEQ ID NO. 31) and R116 (SEQ ID NO. 29) (see figure 1), they were cloned into the RI / HindIII restriction sites in pBS (+) acquired in Stratagene as described above. Variant viruses were cloned using purified viral DNA targets and PCR cloning methods as described in US Patent No. 4,800,159. The resulting plasmids were transformed into the E. coli strain DG101 (ATCC depot No. 47043).

The nucleotide sequences from the 5 'untranslated sequence and the 5' end of the capsid gene of the  isolated prototype C9 and four related isolates, are described and represented below in C9 (SEQ ID NO. 28), R116 (SEQ ID NO. 29), R45 (SEQ ID NO. 30), R110 (SEQ ID NO. 31), and R43 (SEQ ID NO. 32). Sequences depicted do not include KY96 primers (SEQ ID NO. 4), and Ky99 (SEQ ID NO. 25), which were used to facilitate cloning of viral sequences.

C9 - SEQ ID NO. 28

one ACTGTCTTCA CGCAGAAAGC GTCTAGCCAT GGCGTTAGTA TGAGTGTCGT 51 ACAGCCTCCA GGCCCCCCCC TCCCGGGAGA GCCATAGTGG TCTGCGGAAC 101 CGGTGAGTAC ACCGGAATTA CCGGAAAGAC TGGGTCCTTT CTTGGATAAA 151 CCCACTCTGT GTCCGGTCAT TTGGGCGTGC CCCCGCAAGA CTGCTAGCCG 201 AGTAGCGTTG GGTTGCGAAA GGCCTTGTGG TACTGCCTGA TAGGGTGCTT 251 GCGAGTGCCC CGGGAGGTCT CGTAGACCGT GCATCATGAG CACAAATCCT 301 AAACCTCAAA GAAAAACCAA AAGAAACACA AACCGCCGCC CACAGGACGT 351 TAAGTTTCCG GGTGGCGGCC AGATCGTTGG CGGAGTTTAC TTGCTGCCGC 401 GCAGGGGCCC CAGGTTGGGT GTGCGCGCGA CAAGAAAGAC TTCCGAGCGA 451 TCCCAGCCGC GTGGGAGACG CCAGCCCATC CCAAAAGATC GGCGCTCCAC 501 CGGCAAGTCC TGGGGAAAGC CAGGAT

R116 -SEQ ID NO. 29

one GGCGTTAGTA TGAGTGTCGT ACAGCCTCCA GGCCCCCCCC TCCCGGGAGA 51 GCCATAGTGG TCTGCGGAAC CGGTGAGTAC GCCGAATTAC CGGAAAGACT 101 GGGTCCTTTC TTGGATAAAC CCACTCTATG TCCGGTCATT TGGGCGTCCC 151 CCGCAAGACT GCTAGCCGAG TAGCGTTGGG TTGCGAAAGG CCTTGTGGTA 201 CTGCCTGATA GGGTGCTTGC GAGTGCCCCG GGAGGTCTCG TAGACCGTGC 251 ATCATGAGCA CAGATCCTAA ACCTCAAAGA AAAACCAAAA GAAATACAAA 301 CCGCCGCCCA CAGGACGTCA AGTTCCCGGG TGGCGGCCAG ATCGTTGGCG 351 GAGTTTACTT GCTGCCGCGC AGGGGCCCCA GGTTGGGTGT GCGCACAACA 401 AGGAAGACTT CCGAGCGATC CCAGCCGCGT GGAAGACGCC AGCCCATCCC 451 GAAAGATCGG CGCTCCACCG GTAAGTCCTG GGGAAAGCCA GGAT

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R45 - SEQ ID NO. 30

one GGCGTTAGTA TGAGTGTCGT ACAGCCTCCA GGCCCCCCCC TCCCGGGAGA 51 GCCATAGTGG TCTGCGGAAC CGGTGAGTAC GCCGAATTGC CGGAAAGACT 101 GGGTCCTTTC TTGGATAAAC CCACTCTATG TCCGGTCATT TGGGCGTCCC 151 CCGCAAGACT GCTAGCCGAG TAGCGTTGGG TTGCGAAAGG CCTTGTGGTA 201 CTGCCTGATA GGGTGCTTGC GAGTGCCCAG GGAGGTCTCG TAGACCGTGC 251 ATCATGAGCA CAAATCCTAA ACCCCAAAGA AAAACCAAAA GAAACACAAA 301 CCGCCGCCCA CAGGACGTTA AGTTCCCGGG TGGCGGCCAG ATCGTTGGCG 351 GAGTTTACTT GATGCCGCGC AGGGGCCCCA GGTTGGGTGT GCGCGCGACG 401 AGGAAGACTT CCGAGCGATC CCAGCCGCGT GGGAGACGCC AGCCCATCCC 451 GAAAGATCGG CGTTCCACCG GCAAGTCCTG GGGAAAGCCA GGAT

R110 - SEQ ID NO. 31

one GGCGTTAGTA TGAGTGTCGT ACAGCCTCCA GGCCCCCCCC TCCCGGGAGA 51 GCCATAGTGG TCTGCGGAAC CGGTGAGTAC GCCGAATTGC CGGAAAGACT 101 GGGTCCTTTC TTGGATTAAC CCACTCTATG TCCGGTCATT TGGGCGTCCC 151 CCGCAAGACT GCTAGCCTAG TAGCGTTGGG TTGCGAACGG CCTTGTGGTA 201 CTGCCTGATA GGGTGCTTGC GAGTGCCCCG GGAGGTCTCG TAGACCGTGC 251 ATCATGAGCA CAAATCCTAA ACCTCAAAGA AAAACCAAAA GAAACACAAA 301 CCGCCGCCCA CAGGACGTCA AGTTCCCGGG AGGCGGTCAG ATCGTTGGCG 351 GAGTTTACTT GCTGCCGCGC AGGGGCCCCA GGTTGGGTGT GCGCGCGACA 401 AGGAAGACTT CCGAGCGATC CCAGCCGCGT GGGAGACGCC AGCCCATCCC 451 GAAAGATCGG CGCTCCACCG GCAAGTCCTG GGGAAAGCCA GGAT

R43 - SEQ ID NO. 32

one GGCGTTAGTA TGAGTGTCGT ACAGCCTCCA GGCCCCCCCC TCCCGGGAGA 51 GCCATAGTGG TCTGCGGAAC CGGTGAGTAC ACCGAATTAC CGGAAAGACT 101 GGGTCCTTTC TTGGATAAAC CCACTCTATG TCCGGTCATT TGGGCGTCCC 151 CCGCAAGACT GCTAGCCTAG TAGCGTTGGG TTGCGAACGG CCTTGTGGTA 201 CTGCCTGATA GGGTGCTTGC GAGTGCCCCG GGAGGTCTCG TAGACCGTGC 251 ATCATGAGCA CAAATCCTAA ACCTCAAAGA AAAACCAAAA GAAACACAAA 301 CCGCCGCCCA CAGGACGTCA AGTTCCCGGG TGGCGGCCAG ATCGTTGGCG 351 GAGTTTACTT GCTGCCGCGC AGGGGCCCCA GGTTGGGTGT GCGCGCGACA 401 AGGAAGACTT CCGAACGGTC CCAGCCGCGT GGGAGGCGCC AGCCCATCCC 451 AAAAGATCGG CGCTCCACCG GCAAGTCCTG GGGAAAGCCA GGAT

The clones that contained the viral sequences have been deposited in the American Type Culture Collection, Baltimore, Maryland, as follows:

Isolated SEQ ID DO NOT Plasmid Name ATCC deposit nº Date of the Deposit C9 29 pHCV-C9 75265 two July 1992 R110 32 pHCV-R110 75266 two July 1992

Plasmid pHCV-C9 contains the C9 sequence of the new variant. The alignment of the four related variants with the C9 sequences is represented in Figure 1. The sources of the sequence data of the new isolates were as follows: HCV-J1, HCV-J4 (Okamoto et al., 1990, Japan J. Exp. Med. 6 (3): 167-177); HCV-J (Kato et al., 1990, Mol. Biol. Med. 7: 495-501); HCV-BK (Takamizawa et al., 1991, J. Virol. 65 : 1105-1113); and HCV-1 US-PT (Han et al., 1991, Proc. Natl. Acad. Sci. USA 88 : 1711-1715). The numbering is in accordance with Kato et al., 1990, Proc. Natl. Acd. Sci. USA 87 : 9524-9528.

TABLE 3 Nucleotide determination and homology of the amino acid sequence of isolate C9 with the US prototype (PT-HCV) and Japanese isolates (J-HCV)

Sequences Isolated PT-HCV J-HCV 5' UTR Nucleotide (255) C9 92.9% 92.9% J-HCV 98.8% - Core Nucleotide (244) C9 84.9% 85.1% J-HCV 92.1% - Amino acid (80) C9 91.3% 90.0% J-HCV 98.7 -

European Patent No. 92114115.6 provides materials and methods for tests that are specific to the isolated C9 and distinguish the isolated from other isolated from the hepatitis. These methods are based on acid hybridization. nucleic with or without the use of PCR to amplify the target sequences In other versions, the methods employ specific immunoglobulins of isolate C9.

The oligonucleotide sequences of the C9 specific primers and probes are summarized in the Table 4 below.

TABLE 4

Probes SEQ ID DO NOT. 36 MY160 5'-TGTCCGGTCATTTGGGCG-3 ' (nt216-233) I KNOW THAT ID NO. 33 (one) 5'-TTGCCGGAAAGACTGGGTCCTTTC-3 ' (nt174-197) I KNOW THAT ID NO. 3. 4 (two) 5'-CAAAAGAAACACAAACCGCCGCCC-3 ' (nt374-397) I KNOW THAT ID NO. 35 (3) 5'-CCAGCCCATCCCGAAAGATCGGCG-3 ' (nt527-550) Line primers entry SEQ ID NO. 37 (one) 5'-AAACCCACTCTATGTCCGGTC-3 ' (nt204-224) I KNOW THAT ID NO. 38 (two) 5'-GTACGCCGGAATTGCCGGAAA-3 ' (nt163-183) I KNOW THAT ID NO. 39 (3) 5'-CCTCAAAGAAAAACCAAAAGA-3 ' (nt360-380) Line primers exit SEQ ID NO. 40 (4) 5'-TGGCGTCTCCCACGCGGCTGG-3 ' (nt509-529) I KNOW THAT ID NO. 41 (5) 5'-CTTTCCCCAGGACCTGCCGGT-3 ' (nt555-575)

The numbering system used for the Nucleotide identification is described in Kato et al., 1990, Proc. Natl Acad. Sci., See above.

An oligonucleotide, KY150 (SEQ ID NO. 42) is of utility for the detection of both the C9 isolate and the HCV prototype isolates known above. East oligonucleotide has the following sequence:

5'-CATAGTGGTCTGCGGAACCGGTGAGT-3 '

For experts in the specialty it will be evident that any pair of primers of the line of input / output line, indicated in table 4, is suitable for the specific amplification of the C9 isolate and related isotypes. Will be Also evident is the selection of a specific C9 probe suitable for hybridization of the nucleic acid sub sequence amplified using the nucleotide positions as a guide indicated in table 4.

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In a preferred version, the primers of the invention are used in conjunction with an amplification, by polymerase chain reaction (PCR) of nucleic acid Diana. Because HCV is an RNA virus, the first step of the amplification is the synthesis of a copy of the DNA (cDNA) of the region to be amplified. Reverse transcription can be performed as a separate step, or as described below, in a homogeneous reaction of the polymerase chain reaction and reverse transcription (RT-PCR), a modification of the polymerase chain reaction for RNA amplification.

Although the PCR procedure is already well known in the specialty (see U.S. Patent No. 4,683,195; 4,683,202; and 4,965,188), general information is provided below about it for greater clarity and full understanding of the invention for the unfamiliar the PCR procedure.

To amplify a target nucleic acid sequence of a sample, by the PCR procedure, the sequence must be accessible to the components of the amplification system. In general this accessibility is ensured by isolating the nucleic acids from the sample. A wide variety of techniques for extracting ribonucleic acids from biological samples are known in the art. For example, those described in Rotbart et al., 1989, in PCR Technology (Erlich ed., Stockton Press, New York) and Han et al., 1987, Biochemistry 26 : 1617-1625. Alternatively, if the sample is easily disintegrable, the nucleic acid does not need to be purified before amplification with the PCR method, that is, if the sample is composed of cells, in particular lymphocytes or peripheral blood monocytes, lysis and dispersion of the intracellular components can be achieved simply by suspending the cells in a hypotonic buffer.

The first step of each PCR cycle involves the  Duplex nucleic acid separation. Of course, if the acid target nucleic is single stranded, that is, single stranded RNA, no initial separation step is necessary during the first cycle. Once the chains are separated, the next step of the PCR involves hybridization of separate chains with primers that flank the target sequence. The primers extend to continuation to form complementary copies of the chains Diana. For successful PCR amplification, the primers are select so that the position at which each hybrid primer along a duplex sequence is such that the product of extension synthesized from a primer, when separated from the mold (complement), serves as a mold for the extension of the other primer. The cycle of denaturation, hybridization and extension, is Repeat as many times as necessary to get the amount desired amplified nucleic acid.

In the preferred version of the PCR procedure, the separation of the chains is achieved by heating the reaction mixture at a temperature high enough for a time sufficient to cause the denaturation of the duplex, but not to cause a denaturation of the polymerase (see US Pat. No. 4,965,188). A typical heat denaturation implies temperatures ranging from about 80 ° C to 105 ° C for times ranging from seconds to minutes. However, the separation of the chains can also be achieved by any suitable method of denaturation, including physical, chemical or enzymatic means. Chain separation can be induced by a helicase, for example, or an enzyme capable of presenting helicase activity. For example, the enzyme RecA has a helicase activity in the presence of ATP. Suitable reaction conditions for the separation of chains by helicase are already known in the art (see Khun Hoffmann-Berling, 1978, CSH-Quantitative Biology 43 : 63-67; and Radding, 1982, Ann. Rev. Genetics 16 : 405-436).

The template-dependent extension of the PCR primers is catalyzed by a polymerization agent in the presence of suitable amounts of four deoxyribonucleoside triphosphates (typically dATP, dGTP, dCTP and dTTP) in a reaction medium composed of appropriate salts, cations. metallic, and a pH buffer system. Suitable polymerization agents are enzymes already known to catalyze the synthesis of template dependent DNA. Because hepatitis C is an RNA virus, the initial template for primer extension is RNA. They are suitable polymerization agents for the synthesis of a complementary sequence of copy DNA (cDNA), from the RNA template, reverse transcriptase (RT), such as the avian myeloblastosis virus RT, the RT virus Moloney mouse leukemia, or Thermus thermophilus (Tth) DNA polymerase, a thermostable DNA polymerase, with reverse transcriptase activity marketed by Perkin Elmer. Typically, the genomic template of the RNA / cDNA duplex is denatured by heat during the first denaturation one step after the initial reverse transcription step leaving the available DNA chain as an amplification template. Polymerases suitable for use as a DNA template include, for example, E. Coli DNA polymerase I or a Klenow fragment, T4 DNA polymerase, Tth polymerase and Taq polymerase, a thermostable DNA polymerase isolated from Thermus aquaticus and developed and manufactured by Hoffmann-La Roche and marketed by Perkin Elmer. The last enzyme is widely used in the amplification and sequencing of nucleic acids. The reaction conditions for the use of Taq polymerase are already known in the art and are described in Gelfand, 1989, PCR Technology, see above.

When the RNA is amplified, a reverse transcription (RT) step is performed to create a DNA copy (cDNA) of the RNA. Published PCT Patent No. WO 91/09944 published on July 11, 1991, describes a high temperature reverse transcription by means of a thermostable polymerase that also acts in PCR amplification. High temperature RT provides greater specificity and efficiency of the primer. In a `` homogeneous RT-PCR '' the same primers and polymerase are sufficient for both the reverse transcription steps and for the PCR amplification steps, and the reaction conditions are optimized so that both reactions take place at at the same time without a reagent change. Thermus thermophilus DNA polymerase, a thermostable DNA polymerase that can act as a reverse transcriptase, is used for all primer extension steps, regardless of template. The two processes can be carried out without having to open the tube to change or add reagents; Only the temperature profile is adjusted between the first cycle (RNA template) and the rest of the amplification cycles (DNA template).

The 5 'terminal end of the HCV genome is expected to have a significant secondary structure that could inhibit reverse transcription with a transcriptase inverse such as the RT of Moloney mouse leukemia virus, interfering with hybridization of the primer. Unfortunately increasing the reaction temperature to denature the secondary structure, most enzymes of reverse transcriptase. The employment of the activity of the reverse transcriptase of DNA polymerase from Thermus thermophilus recombinant (rTth), allows cDNA synthesis to take place at high temperature without inactivation of enzymes. The primers of the present invention remain hybridized to the RNA template at this high temperature of reverse transcription.

The PCR method can be performed in one way. based on steps, where new steps are added after each step reagents, in a form where all reagents are added simultaneously, or in a form of partial steps, where add fresh or different reagents after a given number of Steps. For example, if string separation is induced by heat and polymerase is heat sensitive, then the polymerase has to be added after each separation cycle of the chain. However, if, for example, a helicase is used for denaturation or if a polymerase is used thermostable for extension, then all reagents can  be added initially or alternatively, if the relationships molars of the reagents are consistent with the reaction, the reagents can be replenished periodically as they go being consumed by the synthesis reaction.

Experts in the specialty already know that the PCR process is performed more commonly as a process automated with a thermostable enzyme. In this process, the reaction mixture temperature follows a cycle through a denaturation region, a region of primer reassociation and a region of the extension reaction. Alternatively, the re-association temperature and extension temperature can be the same. The RT-PCR described in the examples employs this temperature cycling in two steps. A machine specifically adapted for use with a thermostable enzyme It is commercially available.

The experts in the specialty are at current contamination problems of a PCR by acid  amplified nucleic from the amplification of reactions previous and not specific. A method to reduce these problems allows enzymatic degradation of any amplified DNA from previous reactions and reduces amplification not specific. PCR amplification is carried out in the presence of dETP instead of dTTP. The resulting double chain product, which contains uracil is subject to degradation by uracil-N-glucosylase (UNG), while normal thymine-containing DNA is not degraded by the ENG. Adding UNG to the amplification reaction mixture before amplification begins, all the DNA that degrades It contains uracil that could serve as a target. Because the only DNA source containing uracil is the amplified product of An earlier reaction, this method effectively sterilizes the mixture reaction, eliminating the problem of pollution from previous reactions (`` carry over ''). UNG itself becomes temporarily inactive by heat, so that the steps of denaturation of the amplification procedure also serve to inactivate the UNG. Therefore new products of amplification, although uracil is incorporated, in an environment effectively exempt from UNG and do not degrade.

A preferred version of the invention uses a Homogeneous RT / PCR method that incorporates a sterilization step. This non-addition reaction of a tube serves to sterilize the RT-PCR reaction preventing contamination by previous reactions and providing a PCR product detectable amplified from a sample containing an RNA Diana. This method is exemplified in example 6.

Although the preferred version incorporates the RT-PCR amplification, amplification of target sequences of a sample can be performed by any known method, such as ligase chain reaction (CSF), transcription amplification, and self-sustained replication of the sequence, each of which provides enough amplification so that the target sequence can be detected by hybridization of the nucleic acid with an SSO probe. Alternatively, methods that amplify the probe to detectable levels, such as amplification of the Qβ-replicase. The term `` probe '' covers the oligonucleotides of the specific sequence used in the previous procedures; for example, the two or more oligonucleotides used in CSF are `` probes '' for purposes of the present invention, although some versions of the CSFs require ligation of probes to indicate presence of an allele.

Hybridization of probes of a sequence specific is an important step for the successful completion of The present methods. The oligonucleotide probes of a specific sequence of the present invention hybridize specifically with a particular segment of the HCV genome and they have destabilizing mismatches with the sequences of other organisms In hybridization conditions sufficiently strict, the probes hybridize specifically only in sequences exactly complementary. The strictness of the conditions of hybridization can relax to tolerate varying amounts of sequence mismatches. Product detection amplified uses this hybridization of the specific sequences to ensure detection of only the amplified target correct, thereby decreasing the possibility of a false positive caused by the presence of homologous sequences from of related organisms.

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The analysis methods to detect hybrids formed between SSO probes and nucleic acid sequences can require that the probes have additional features in addition from the hybridization region. For example, if first the probe is immobilized, as in the `` dot blot '' format (`` point transfer '') `` reverse '' described more forward, the probe may also contain long stretches of poly-dT that can be attached to a nylon support by  irradiation, a technique described in more detail in the Patent PCT publication nº 89/11548. In the `` dot blot '' format, the target immobilized hybridizes with probes containing a compound used in the detection procedure, as described more ahead.

The probes of the invention can be synthesized and labeled using the techniques described above for oligonucleotide synthesis. The probe can be labeled at the 5 'end with 32 P by incubating the probe with 32 P-ATP and kinase. A suitable non-radioactive brand for SSO probes is horseradish peroxidase (HRP). The methods for preparing and detecting the detection probes containing this mark are described in the examples below and in US Patent Nos. 4,914,210 and 4,962,029. For additional information on the use of such labeled probes, see US Patent No. 4,789,630; Saiki et al., 1988, N. Eng. J. Med. 319 : 537-541; and Bugawan et al., 1988, Bio / Technology 6 : 693-947. The use of chromogens include leuco red dye and 3,3 ', 5,5'-tetramethyl-benzidine (TMB).

The probes of the invention can be used to determine if viral sequences are present in a sample, determining if SSO probes bind to sequences virals present in the sample. Analysis methods appropriate to the purposes of the present invention to detect hybrids formed between the SSO probes and the nucleic acid sequences in A sample, are already known in the specialty. For example, the detection can be performed using a dot blot, such as It is described in the examples. In the `` dot blot '' format, the sample  amplified that has not been labeled binds to a solid support, such as a membrane, the membrane is incubated with the sample marked in suitable hybridization conditions, the unhybridized probe will washed away, and the filter is controlled to detect the presence of attached probe. When multiple samples are analyzed with a single probe, the `` dot blot '' format is perfectly useful. Many samples can be immobilized in discrete positions on a single membrane and hybridize simultaneously submerging the membrane in a probe solution.

An alternative method that is perfectly useful when a large number of different probes must be used, it is a formed `` dot blot '' `` inverse '', in which the sequence amplified contains a mark, and the probe is attached to the support solid. This format would be useful if the essay of the present invention will be used as a test of a battery of tests to perform simultaneously. In this format, SSO probes without mark are attached to the membrane and exposed to the sample marked in appropriate strict conditions of hybridization. Then it Remove the marked sample without hybridizing, washing under conditions strict strict, and the filter is then controlled to Detect the presence of linked sequences.

Both direct and dot tests blot '' inverses can be conveniently performed on a plate microtiter The probes can be fixed to serum albumin bovine (BSA), for example, which adheres to the plate of microtiter, whereby the probe is immobilized.

In another suitable test system, a probe Marking is added during the PCR amplification process. Any SSO probe that hybridizes with a target DNA during each step of synthesis is degraded by the 5 'to 3' exonuclease activity of a polymerase, p. ex. Taq polymerase. Then it detects the degradation product of the probe. In this way, the presence of a degraded product indicates that hybridization between the SSO probe and the target DNA has taken place.

The nucleotide sequences obtained formerly they constitute an important aspect of the present invention. Although only one string of the sequence is shown, experts in the specialty recognize that the other chain of the sequence can be deduced from the information represented previously. This information allows the construction of others probes and primers of the invention.

The present invention also relates to kits, multi-container units containing useful components to carry out the present method. A useful kit may contain SSO probes to detect the nucleic acid of the virus hepatitis C. In some cases the SSO probes can be fixed to a adequate support membrane. The kit also contains primers for RT-PCR, since such primers are useful in the preferred version of the invention. Other useful kit components include, for example, reverse transcriptase or polymerase, the nucleoside triphosphate substrate, the means used for the labeled (for example, an avidin-enzyme conjugate, and an enzyme and chromogen substrate if the brand is biotin), and buffers suitable for reverse transcription, PCR or hybridization reactions. In addition to the previous components, the kit may also contain instructions to perform the method in question.

Example 1

In homogeneous RT-PCR, the same Polymerase acts at the same time as reverse transcriptase and DNA polymerase This allows both the initial reverse transcription of the HCV genomic RNA as the subsequent amplification of the cDNA that they are made in the same tube without having to open the tube between the different steps to change the reagents.

Nucleic acids were isolated from serum or plasma using the IsoQuick ™ nucleic acid extraction kit of MicroProbe. All amplifications were performed using a TC9600 Thermocycler ™ and MicroAmp ™ tubes, both from Perkin Elmer, in total reaction volumes of 20 µl. Reagents for each 20 µl reaction are summarized in table 5, a continuation:

TABLE 5

Reaction mixture RT-PCR H2O 6.3 \ mul 10X RT reaction buffer: (100 mM Tris-HCl (pH 8.3), 900 mM KCl) 2.0 \ mul MnCl2 (10 mM, 0.85 mM concentration final) 1.7 \ mul DNTP (2 mM each of dATP, dCTP, dGTP and DTTP in H2O, pH 7.0) 2.0 \ mul Primer KY78 (SEQ ID NO. 18) (1.5 µM in H2O, 3 picomoles / final reaction concentration) 2.0 \ mul KY80 primer (SEQ ID NO. 5) (1.5 µM in H2O, 3 picomoles / final reaction concentration) 2.0 \ mul RTth DNA polymerase (Perkin Elmer, Norwalk),   CT, USA; 0.18 µM or 2.5 units / µlen IX enzyme storage buffer (20 µM Tris-HCl (pH 7.5), 100 mM KCl, EDTA 0.1 µM 1 µM DTT, Tween ™ 20 (Pierce Surfactants) 0.2%, glycerin 50% [v / v]) 2.0 \ mul Mold nucleic acid (<250 ng total in dH 2 O) 2.0 \ mul

The TC9600 was programmed with the following profile of temperature: after preheating at 70 ° C for 1 minute, it interrupted the program long enough to enter the reaction tubes, the program was restarted, and the temperature at 70 ° C for 15 minutes to allow reverse transcription. Then the reaction temperature is  increased to 95 ° C for 1 minute to denature the RNA duplex - CDNA Then the reaction temperature was subjected to two 95 ° C cycles for 15 seconds and 60 ° C for 20 seconds, followed by 38 cycles of 90 ° C for 15 seconds and 60 ° C for 20 seconds Finally, the reaction temperature was maintained at 60 ° C for 4 minutes for the final extension step, cooled to 15 ° C, and was maintained at this temperature until the analysis of the amplified sample could be performed.

Example 2

Using the detection method with the formed `` dot blot '', a small portion of the AND is denatured amplified and applied to a nylon filter, which hybridizes to then with a marked probe. The hybridization region of the probe is the KY88 (SEQ ID NO. 12), and the probe is marked radioactively with 32 P. Alternatively, the probe can conjugate covalently with horseradish peroxidase (HRP) to obtain a non-isotopic detection means in the presence of a chromogenic or chemiluminescent substrate.

The amplification reaction is carried out generally as described in example 1. Next, PCR amplified product is denatured by means of a alkali treatment. To 5 µl of the PCR product are added 5 µL of 0.5M EDTA, pH 8.0, 8 µL of 5N NaOH, and 82 µl of H2O. The mixture is allowed to stand at room temperature for 10 minutes to complete the denaturation.

BioDyne ™ B (Pall) nylon filters are prepared Corp., Glen Cove, NY, USA), immersing them in H2O for 5 to 10 minutes and then washed with 200 µL of H2O after that the multiple `` dot blot '' (Bio-Dot ™ from Bio Rad, Richmond, CA, USA) is ready. After denaturation, are applied under vacuum, 100 µl of the sample mixture to the Nylon membrane using the dot blot device. TO then rinse each well with 200 µL of 0.4 N NaoH and the complete filter is rinsed briefly with 2X SSC, and dried at air until there are no traces of liquid left. DNA is immobilized and it is reticulated with the nylon filter by ultraviolet radiation with a flow of 1200 mJ / cm2 with a UV illumination box Stratalinker ™ (Stratagene, La Jolla, CA, USA) (setting of `` self-cross-linking '').

The filters are `` pre-hybridized '' immersing them in hybridization buffer (5X SSPE, 5X solution of Denhardt, 0.1% SDS, 50 µg / ml herring sperm DNA) in heat sealable bags at 50 ° C (air stirrer) for At least 30 minutes. The buffer is then replaced with a equal amount of the same solution containing 1-2 x 10 5 cpm / ml probe, and the filter is allowed to hybridize between 2 hours and overnight at 50 ° C.

After hybridization the filters are washed three times in 2X SSPE / 0.1% SDS; twice for 20 minutes at room temperature, and then once for twenty minutes at the highly strict temperature of 60 ° C. Following, the filters are dried with blotting paper, covered with a cover of plastic and exposed to an X-ray film at -70 ° C with one or Two intensification screens.

An alternative method of visualization is to hybridize with oligonucleotide probes conjugated with horseradish peroxidase, prepared as described by Levenson and Chang, 1989, in `` PCR Protocols: A Guide to Methods and Applications ''. PRC: A guide to methods and applications '') (Innis et al., Eds., Academic Press, San Diego), p. 92-112, and Saiki et al., 1988, N. Eng. J. Med. 319 : 537-541, each of which is incorporated by reference. Hybridization is performed with 2 pmoles of HRP-SSO probe per 5 ml of hybridization solution.

After washing, the filters that are going to revealed with a substrate of a chromogenic dye, rinsed with 100 mM sodium citrate, pH 5.0, then placed in 100 mM sodium citrate, pH 5.0 containing 0.1 mg / ml of 3,3 ', 5,5'-tetramethylbenzidine per milliliter (Fluka), and 0.0015 percent hydrogen peroxide, and incubated with a gentle stirring for 10 to 30 minutes at room temperature. The revealed filters are rinsed in water and photographed immediately. The TMB detection system is prepared and employs substantially as described in the typing kit of AmpliType ™ DQα DNA marketed by Perkin Elmer. In another version, the filters are revealed with the detection system chemo-luminescent (ECL; Amersham, Arlington Heights, IL, USA). The filters are rinsed in PBS for 5 minutes and placed in the ECL solution for 1 minute with gentle agitation. The filters are then exposed to a film of X-rays at room temperature for 1 to 5 minutes.

Example 3

In this version of the invention the reverse dot blot format is used. The hybridization region of the probe is KY88 (SEQ ID NO. 12), KY150 (SEQ ID NO. 42) or MY160 (SEQ ID NO. 36), and the probe is fixed to a membrane. The amplified target DNA is hybridized with the probe previously bound to the membrane, as described in Saiki et al., 1989, Proc. Natl. Acad. Sci. 86 : 6230-6234; and in the product insert of the AND AmpliType® DQalpha typing kit, marketed by Perkin Elmer. The amplification primers are biotinized, as described by Levenson and Chang, 1989, see above, so that any amplified DNA that hybridizes with membrane bound probes can be easily detected.

In one version, the detection is carried out by the reaction of horseradish peroxidase with streptavidin (SA-HRP) with any biotinylated amplified DNA, hybridized with the membrane bound probe. In this way, the HRP binds through the SA-biotin interaction, to the DNA  amplified and can be used to generate a signal by means of a wide variety of known media, such as p. ex. the generation of a compound colored by coloring the tetramethylbenzidine (see U.S. Patent No. 4,789,630).

Although the probe can be fixed to the membrane by any means, a preferred method is to `` add a tail '' to the hybridization region of the probe with a sequence much longer poly-dT. The resulting `` tail '' of poly-dT can then react with amino groups of a nylon membrane to fix the probe covalently to the membrane. This reaction can be facilitated. by UV irradiation.

Terminal deoxyribonucleotidyl transferase (TdT, Ratliff Biochemicals; for previous reactions presupposes a concentration of approximately 120 units / mul, which is 100 pmoles / \ mul), can be used to create a queue of poly-dT, although the probe can also be synthesized with tail in a commercially available synthesizer. When use a DNA synthesizer to build the probe with tail, however, the tail is placed at the 5 'end of the probe, of way that premature termination of the chain, unwanted, has place first in the tail region.

TdT reactions should be carried out in a volume of approximately 100 µl containing 1X TdT of salts, 200 pmoles of oligonucleotide, DTT 800, and 60 units of Tdt. 10X TdT of salts consists of 1,000 mM K-cacodylate, 10 mM CoCl 2, 2 mM dithiothreitol, 250 mM Tris-Cl, pH 7.6 and is prepared as described by Roychoudhury and Wu, Meth. Enzymol 65 : 43-62. If convenient, a 10X stock solution of 8 mM dTTP (neutralized to pH 7 with NaOH) can be prepared.

The TdT reaction should be carried out at 37 ° C for two hours and then interrupted by addition of 100 µL of 10 mM EDTA, pH 8. The final concentration of the tail oligonucleotide is 1 µM (1 pmol / µl), and the tail length of homopolymer is approximately 400 remains. The tail length can be changed by adjusting the ratio  from dTTP to oligonucleotide. Tail probes can be stored at -20ºC until the moment of use.

The preferred nylon membrane for the format of Inverse `` dot blot '' is the 0.45 Biodyne ™ B nylon membrane micron pore size, manufactured by Pall and marketed also by ICN as a BioTrans ™ nylon membrane. The probes can apply as spots on the membrane very conveniently with the device for `` dot blot '' Bio-Dot ™ manufactured by BioRad. Each probe is applied stain-shaped in a unique discrete situation in the membrane. Approximately 2 to 10 picomoles of each probe with tail is mixed previously with 50 to 100 µl of TE buffer before application to the `` dot blot '' device. Once the `` dot blot '' has been completed, place the membrane briefly on absorbent paper to separate excess liquid. Then the membrane is placed inside a lighting box, such as p. ex. The box of Stratalinker ™ lighting manufactured by Stratagene, and is exposed from 50 to 60 millijoules / cm2 flow at 254 nm to fix the probe with nylon membrane glue. After a brief rinse for about 15 minutes with hybridization solution to  remove the unbound probe, the membrane is ready for hybridization with the PCR product, biotinylated.

The products of PCR amplification, are denatured by heating at 95 ° C for 3 to 10 minutes and add 40 µl of the denatured PCR product to each probe panel for hybridization. Hybridization is performed at 57 ° C for 20 minutes in a water bath with stirring in a buffer of hybridization composed of 0.5X SSPE, 0.25% SDS, and 5X of Denhardt solution (20X SSPE contains 0.02 EDTA, 0.2M NaH2PO4, 3.6M NaCl, 0.11M NaOH, adjusted to pH 7.4). The hybridization buffer is replaced with 3 ml of a solution that consists of 25 µL of SA-HRP in 3.1 ml of buffer hybridization, and incubate for 20 minutes at 57 ° C in a bath Mary with agitation.

Washing is done in a 2X wash buffer SSPE and 0.1% SDS. After a brief rinse of the membrane in 10 ml of wash buffer, a strict wash of 12 is performed minutes in 10 ml of buffer, at 57 ° C. Then another one is made 5 minute wash at room temperature, followed by washing 5 minutes in 10 ml of 0.1M sodium citrate, pH 5.0.

The binding of the chromogen is carried out in 5 ml of chromogenic solution consisting of 5 ml of 0.1M sodium citrate, 5 µl of 3% hydrogen peroxide and 0.25 ml of chromogen (TMB of Perkin Elmer) for 25-30 minutes at temperature environment. Three 10-minute washes were performed in distilled water at room temperature. A final wash of 1X PBS at temperature environment for 30 minutes can generate a quality signal. During these steps that include the chromogen, the membrane must Protect from light by an aluminum foil cover. The revealed membrane must be photographed to have a record permanent of it.

Example 4

In this version of the invention the microtiter plate format. The probe is fixed in a well of a microtiter plate. The amplified target DNA hybridize with the probe attached as described above. How In the preceding example, the amplification primers are biotinan to allow the detection of amplified DNA, which hybrid with the probes fixed.

First, the desired conjugate probes with BSA they are allowed to be absorbed by the plastic surface of the individual wells. Then these wells are block with a protein like p. ex. bovine serum albumin. From preferably 96-well microtiter plates are used, available at Corning.

Once the amplification is complete, the PCR tubes are taken from the thermal cycler (available from the firm) Perkin Elmer). One hundred microliters of solution are added denaturation to each PCR tube. A new tip of Pipette for each tube. In one version, the detection could not be done immediately. In this case, the PCR tubes are kept overnight at 2 ° C to 8 ° C. The reactions of denatured amplification became viscous after a storage from 2ºC to 8ºC. The tubes were heated briefly of 25ºC to 30ºC before opening the tubes for easy use the pipette

An appropriate number of eight strips were removed of microtiter plate wells (at least 2 strips) and were placed in the frame of the microtiter plate. I know pipetted one hundred microliters of hybridization buffer in each microtiter plate well.

Denaturation solution contains NaOH 0.4M; 80 mM EDTA and 0.005% thymol blue. The buffer of hybridization / neutralization contains: 2.5M NaSCN; NaH_ {PO} {4} 80 mM; 10 mM NH 2 PO 4; and Tween ™ 20 0.125%. The pH before use, which should be 5.0 ± 0.2.

Using tips inserted with a pipettor multichannel, 25 µL amplification reaction was pipetted denatured, from each PCR tube from the cuvette to the corresponding position of the microtiter plate well. The plate was covered with the microtiter plate lid and was tapped 10 to 15 times on the side of the plate. The wells in which the correct reagent had been pipetted, they turned a light yellow color. In those in which it was not observed no change or just a slight change in the color blue, it added an excess of amplicon. The trial is continued when they increase positive OD values, but negative OD values influence. The plate was incubated for 60 minutes at 37 ° C.

After incubation, the plate was washed five times with wash solution. The plate wash must be done manually or with an automatic plate washer microtiter properly programmed. For washing, it is used a 1X wash buffer for PCR. A concentrated wash buffer 10X PCR, was prepared as follows: 9.94 grams per liter of phosphate monosodium (dibasic); 4.41 grams per liter of disodium phosphate (monobasic); 3,722 grams per liter of EDTA; 87.66 grams per liter of sodium chloride; 13.7 grams per liter of Tween ™ 20; and 10 grams per liter of Pro Clin 300 (Rohm and Haas, Philadelphia, PA). The pH of the solution is adjusted with phosphoric acid (a pH 6.5-7.1).

For manual washing the contents of the plate are He emptied and shook himself with light blows until dry. They were added three hundred microliters of wash solution to each well of the plate that was analyzed, and the plate was allowed to dry for 15 to 30 seconds. The plate emptied again and shook lightly blows to dryness. This washing process was repeated four more times.

For an automatic plate washer microtiter, the following procedure was used. He sucked the contents of the wells. The washer was programmed to add 350 microliters of working wash solution to each well of the plate that was analyzed was submerged for 30 seconds and He inhaled. The steps were repeated four more times. Then you He shook the plate with slight blows to dryness.

One hundred microliters of conjugate was added to each  well of the plate being analyzed. The conjugate Avidin-HRP is prepared as follows. Thinner contains 0.1 molar; 0.25% of Emulsit 25 (DKS International, Inc., Tokyo Japan); 1.0% of Kathon CG (Rohm and Hass, Philadelphia, PA); 0.1% phenol; 1.0% bovine globulinagamma. The solution was adjusted  at pH 7.3 with concentrated HCl. To this diluent 10 nM were added of conjugated avidin (Vector Labs, Burlingame, CA). Then the plate was covered and incubated 50 minutes at 37 ° C and washed again as described above. The work substrate was prepared mixing 2.0 ml of substrate. A and 0.5ml of substrate B for each multiple of two strips of 8-well microtiter plates (16 trials). Substrate A contains 3 hydrogen peroxide; 6.5 mM citrate and 0.1% Kathon CG. Substrate B contains 4.2 mM of 3.3 ', 5.5' tetramethylbenzidine and 40% dimethylformamide. The Work substrate was prepared no more than three hours before its use and stored protected from sunlight.

One hundred microliters of substrate of work (mixture of substrates A and B) to each well of the plate that  It's analized. The plate was then covered and incubated in the dark for 10 minutes at room temperature (from 20 ° C to 25 ° C). One hundred microliters of the interruption reagent was added (5% H 2 SO 4) to each well that is analyzed. The absorbance of each well of 450 mM after one hour later of adding the interruption reagent. The absorbance value It was registered for the sample and as a control.

Example 5

The oligonucleotides of the present invention they provide numerous combinations of primers and probes that can be used in the amplification of genomic sequences of the HCV, and for the detection of the amplified product. It has been analyzed a large number of oligonucleotides to determine its effectiveness as primers in an RT-PCR amplification.

PCR amplification reactions are performed with several pairs of oligonucleotides using the methods described in example 1. The amplified product is detected by standard methods of separation and staining by agarose gel electrophoresis (Sambrock et al., see more above). Each of the KY65 input line primers (SEQ ID DO NOT. 1) and KY98 (SEQ ID NO. 3) acted effectively with each of the Starting line primers KY68 (SEQ ID NO. 26), KY95 (SEQ ID NO. 20), and KY99 (SEQ ID NO. 25), in the amplification sequences of the HCV The KY67 (SEQ ID NO. 10) acted effectively with both the KY68 (SEQ ID NO. 26) as with KY99 (SEQ ID NO. 25). Each of the input line primers KY80 (SEQ ID NO. 5), KY83 (SEQ ID NO. 7), KY84 (SEQ ID NO. 8), KY85 (SEQ ID NO. 9), and KY144 (SEQ ID NO. 6) acted effectively with each of the line primers of exit KY78 (SEQ ID NO. 18), KY95 (SEQ ID NO. 20), KY145 (SEQ ID DO NOT. 19), KY148 (SEQ ID NO. 17), and KY149 (SEQ ID NO. 16). Finally each of the KY80 input line primers (SEQ ID NO. 5), and KY81 (SEQ ID NO. 11) acted effectively with the KY100 (SEQ ID DO NOT. twenty-one).

The amplification amplification product PCR using KY78 (SEQ ID NO. 18), KY80 (SEQ ID NO. 5) as primers were detected by probing with each of the KY88 (SEQ ID NO. 12), KY84 (SEQ ID NO. 8), and KY85 (SEQ ID NO.9), using the methods described in example 2, see more above.

Primers KY153 (SEQ ID NO. 15), KY159 (SEQ ID NO. 16), and KY148 (SEQ ID NO. 17), cannot be used with none of the input line primers that are line of Departure from KY85. The 3 'end of KY78 (SEQ ID NO. 18) is adjacent to the 3 'end of KY88 (SEQ ID NO. 12), so although these two primers could function as a primer pair, the lack of a Separator sequence makes independent probing impossible. The primer KY85 (SEQ ID NO. 13) can only be paired with the KY100 (SEQ ID NO. 21), KY99 (SEQ ID NO. 25), KY109 (SEQ ID NO. 23) and KY111 (SEQ ID NO. 24). The KY87 primer (SEQ ID NO. 14) can pair only with KY99 (SEQ ID NO. 25), KY109 (SEQ ID DO NOT. 23) and KY111 (SEQ ID NO.24).

The KY84 (SEQ ID NO. 8), KY85 (SEQ ID NO. 9), KY87 (SEQ ID NO. 14), and KY148 (SEQ ID NO. 17), described above they are useful for the detection of known HCV isolates other than isolated C9. The probes and primers summarized in the Table 3 are specific for the extension and detection of -C9 and related variants and for the exclusion of HCV prototypes from Japan and U.S.

Examples of primers for amplification are Specific prototypes of HCV from Japan and U.S. and not of HCV-C9, the following: KY84 (SEQ ID NO. 8), KY85 (SEQ ID NO. 9), KY148 (SEQ ID NO. 17) and KY87 (SEQ ID NO. 14). The primers for the amplification of HCV prototypes from Japan, U.S. and C9, include KY67 (SEQ ID NO. 10), KY78 (SEQ ID NO. 18), KY80 (SEQ ID NO. 5), KY81 (SEQ ID NO. 11), and KY83 (SEQ ID NO. 7), KY86 (SEQ ID NO. 13), KY88 (SEQ ID NO 12), and KY95 (SEQ ID NO. 20), KY1000 (SEQ ID NO. 21), KY144 (SEQ ID NO. 6) and KY145 (SEQ ID NO. 19), and KY153 (SEQ ID NO. 15). The KY65 primers (SEQ ID NO. 1), and KY68 (SEQ ID NO. 27), KY98 (SEQ ID NO. 3), KY99 (SEQ ID NO.25), KY109 (SEQ ID NO. 23), KY111 (SEQ ID NO. 24) and KY149 (SEQ ID NO. 16), are suitable for amplification of HCV prototypes from Japan and U.S. and isolated related variants, and can be also suitable for the detection of HCV-C9 e related isotypes.

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Examples of probes for the detection of HCV prototypes from Japan, U.S. and C9 and related variants, the following: KY67 (SEQ ID NO. 19), KY78 (SEQ ID NO. 18), KY81 (SEQ ID NO. 11), KY86 (SEQ ID NO. 13), KY95 (SEQ ID NO. 20), KY150 (SEQ ID NO. 42), and KY145 (SEQ ID NO. 19). The probes for detection of the HCV prototype of Japan and U.S., and related variants, the which probes do not detect the prototype of HCV-C9 include KY83 (SEQ ID NO. 7), KY87 (SEQ ID NO. 14), KY84 (SEQ ID NO. 8), KY88 (SEQ ID NO. 12), KY85 (SEQ ID NO. 9), KY100 (SEQ ID DO NOT. 21), KY148 (SEQ ID NO. 17) and KY149 (SEQ ID NO. 16). Probes KY65 (SEQ ID NO. 1), KY68 (SEQ ID NO. 26), KY82 (SEQ ID NO. 27), KY99 (SEQ ID NO. 25), KY109 (SEQ ID NO. 23), and KY111 (SEQ ID NO. 24), KY80 (SEQ ID NO. 5), KY144 (SEQ ID NO. 6), and KY153 (SEQ ID NO. 15), are suitable for the detection of the HCV prototype of Japan and U.S. and isolated related variants and may also be suitable for the detection of HCV-C9 and related variants.

Example 6

A preferred method is RT / PCR amplification. homogeneous HCV RNA in the presence of UNG. The method of homogeneous RT-PCR amplification described in the Example 1, was modified to include a sterilization step. The protocol described below demonstrates that RT reactions and PCR incorporate dUTP. Sterilization takes place at 50 ° C before of the passage of the RT. At the high reaction temperatures of the RT-PCR, the UNG is inactive, and the products that contain dU are not degraded. Two units of UNG (Perkin Elmer) successfully sterilized a contamination equivalent to 0.25 µm of a 100 µl amplification made with 10,000 copies of RNA. Higher amounts of UNG, for example, up to 6 units per 100 mul reaction are also suitable.

The components of the mixture of reaction in the following order:

\ mul / Rx 50% glycerin 16.00 16.00 10X RT Rxn. Tampon (100 mM Tris-HCl (pH 8.3) KCl 900 mM) 10.00 DGTP (10 mM); 200 µM final 2.00 DATP (10 mM); 200 µM final 2.00 DUTP (20 mM); 200 µM final 1.00 DCTP (10 mM); 200 µM final 2.00 KY80 (SEQ ID NO. 5) at 15 µM (15 pmoles each / rxn final); (+) string primer biotinylated 1.00 KY78 (SEQ ID NO. 18) to 15 µM (15 pmoles each / rxn final); (-) biotinyl chain, primer RT 1.00 UNG: 1 unit / \ mul 2.00 RTth DNA polymerase: 2.5 U / mul in enzyme storage buffer 1X * 4.00 MnCl2 (10 mM); 0.9 mM final   9.00 Mother Mix by tube 50.00 RNA (with support ** background in H2O)  50.00 Total volume of reaction 100.00 * 1X buffer storage enzyme = (Tris-HCl [pH 7.5] 20 mM, 100 mM KCl, EDTA 0.1 mM, DTT 1 mM, 0.5% Tween ™ 20 [Pierce Surfactamps], 50% glycerin [v / v]). ** 1 mg of RNA homopolymeric poly-rA, from Pharmacia (the poly-rA No. 27 has an average S 20, w} of 8.8 (margin 6- 13) or some non-specific products at the temperature of reassociation employees at entry levels of less of 200 ng per reaction. They have examined the calf thymus DNA, PBL DNA, placenta DNA human and DNA of the K562 cell line, and behave in a way Similary.

Process

one.

Connect the TC9600 thermal cycler to preheat the cover.

two.

Prepare the mixture of reaction at room temperature.

3.

Place the tubes on the thermal cycler and press `` Run '' to restart The thermal cycler.

Four.

Start up the machine in signal no. 8.

5.

Terms thermocycling requested.

Signal 1: Keep 2 minutes at 50 ° C (sterilization step (UNG)).

Sign 2: Keep 15 minutes at 70 ° C (reverse transcription step).

Signal 3: Keep 1 minute at 95 ° C.

Sign 4: Two temp. PCR 15 sec. at 95 ° C and 10-30 sec. at 60 ° C for 2 cycles

Sign 5: Two temp. PCR 15 sec. at 90 ° C and 10-30 sec. at 60 ° C for 38 cycles

Sign 6: Hold at 72 ° C for 1 hour.

Signal 7: Hold at 15 ° C without define.

Signal 8: Link signals: 1, 2, 3, 4, 5, 6 and 7.

In step 5, signal 2, the 15 minute time of  reaction can be lowered to 5 minutes without decreasing efficiency of the reaction. In step 5, signal 5, for long G + C molds, it may be preferable to increase the denaturation temperature to 95 ° C The analysis of the reaction products by Assay format with microtiter plate, gave usually as a result higher absorbance values of 0.8 for positive samples and = 0.5 for negative samples.

(1. GENERAL INFORMATION:

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CANDIDATE:

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NAME: F. Hoffmann-La Roche Ltd.

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STREET: Grenzacherstrase 124

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CITY: Basel

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STATE: BS

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COUNTRY: Switzerland

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POSTAL CODE (ZIP): CH-4070

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FACSIMILE: (0) 61 688 13 95

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TELEX: 962292/965512 hlc ch

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TITLE OF INVENT: Primers and probes for hepatitis detection C.

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(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 30 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 13:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

GGTGCTTGCG AGTGCCCCGG GAGGTCTCGT

\hfill

30

 \ dddseqskip 

GGTGCTTGCG AGTGCCCCGG GAGGTCTCGT

 \ hfill 

30

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 14:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 24 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 14:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

GACTTCCGAG CGGTCGCAAC CTCG

\hfill

24

 \ dddseqskip 

GACTTCCGAG CGGTCGCAAC CTCG

 \ hfill 

24

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 15:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 25 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 15:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CCCAACATA CTCGGCTAGC AGTCT

\hfill

25

 \ dddseqskip 

CCCAACATA CTCGGCTAGC AGTCT

 \ hfill 

25

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 16:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 26 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 16:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

AAGGCCTTTC GCGACCCAAC ACTACT

\hfill

26

 \ dddseqskip 

AAGGCCTTTC GCGACCCAAC ACTACT

 \ hfill 

26

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 17:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 26 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 17:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CACAAGGCCT TTCGCGACCC AACACT

\hfill

26

 \ dddseqskip 

CACAAGGCCT TTCGCGACCC AACACT

 \ hfill 

26

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 18:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 24 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 18:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CTCGCAAGCA CCCTATCAGG CAGT

\hfill

24

 \ dddseqskip 

CTCGCAAGCA CCCTATCAGG CAGT

 \ hfill 

24

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 19:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 26 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 19:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CACTCGCAAG CACCCTATCA GGCAGT

\hfill

26

 \ dddseqskip 

CACTCGCAAG CACCCTATCA GGCAGT

 \ hfill 

26

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 20:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 29 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 20:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

GGGAATTCGC AAGCACCCTA TCAGGCAGT

\hfill

29

 \ dddseqskip 

GGGAATTCGC AAGCACCCTA TCAGGCAGT

 \ hfill 

29

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 21:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 23 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 21:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CGAGGTTGCG ACCGCTCGGA AGT

\hfill

 \ dddseqskip 

CGAGGTTGCG ACCGCTCGGA AGT

 \ hfill 

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 22:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 21 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 22:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

AGGTGCGAC CGCTCGGAAG T

\hfill

21

 \ dddseqskip 

AGGTGCGAC CGCTCGGAAG T

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 23:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 21 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 23:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

AATGCCATAG AGGGGCAAG G

\hfill

21

 \ dddseqskip 

AATGCCATAG AGGGGCAAG G

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 24:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 21 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 24:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

ATTGCCATAG AGGGGCAAG G

\hfill

21

 \ dddseqskip 

ATTGCCATAG AGGGGCAAG G

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 25:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 31 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 25:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CAGAATTCAT TGCCATAGAG GGGCCAAGGA T

\hfill

31

 \ dddseqskip 

CAGAATTCAT TGCCATAGAG GGGCCAAGGA T

 \ hfill 

31

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 26:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 22 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 26:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CAGAATTCGC CCTCATTGCC AT

\hfill

22

 \ dddseqskip 

CAGAATTCGC CCTCATTGCC AT

 \ hfill 

22

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 27:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 24 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 27:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CCCACCCCAA GCCCTCATTG CCAT

\hfill

 \ dddseqskip 

CCCACCCCAA GCCCTCATTG CCAT

 \ hfill 

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 28:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 526 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 28:

one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 29:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 494 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 29:

9

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 30:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 494 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 30:

10

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 31:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 494 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 31:

eleven

12

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 32:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 494 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 32:

13

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 33:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 24 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 33:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

TTGCCGGAAA GACTGGGTCC TTTC

\hfill

24

 \ dddseqskip 

TTGCCGGAAA GACTGGGTCC TTTC

 \ hfill 

24

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 34:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 24 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 34:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CAAAAGAAAC ACAAACCGCC GCCC

\hfill

24

 \ dddseqskip 

CAAAAGAAAC ACAAACCGCC GCCC

 \ hfill 

24

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 35:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 24 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 35:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CCAGCCCATC CCGAAAGATC GGCG

\hfill

24

 \ dddseqskip 

CCAGCCCATC CCGAAAGATC GGCG

 \ hfill 

24

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 36:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 30 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 36:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

TGTCCGGTCA TTTGGGCG

\hfill

18

 \ dddseqskip 

TGTCCGGTCA TTTGGGCG

 \ hfill 

18

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 37:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 30 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 37:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

AAACCCACTC TATGTCCGGT C

\hfill

21

 \ dddseqskip 

AAACCCACTC TATGTCCGGT C

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 38:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 21 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 38:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

GTACGCCGGA ATTGCCGGAA A

\hfill

21

 \ dddseqskip 

GTACGCCGGA ATTGCCGGAA A

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 39:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 21 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 39:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CCTCAAAGAA AAACCAAAAG A

\hfill

21

 \ dddseqskip 

CCTCAAAGAA AAACCAAAAG A

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 40:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 21 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 40:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

TGGCGTCTCC CACGCGGCTG G

\hfill

21

 \ dddseqskip 

TGGCGTCTCC CACGCGGCTG G

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 41:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 21 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 41:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CTTTCCCCAG GACCTGCCGG T

\hfill

21

 \ dddseqskip 

CTTTCCCCAG GACCTGCCGG T

 \ hfill 

twenty-one

           \ vskip0.666000 \ baselineskip
        

(2) INFORMATION FOR SEQ ID No. 42:

           \ vskip0.666000 \ baselineskip
        

(i)

CHARACTERISTICS OF SEQUENCE:

           \ vskip0.333000 \ baselineskip
        

(TO)

LENGTH: 26 base pairs

           \ vskip0.333000 \ baselineskip
        

(B)

TYPE: acid nucleic

           \ vskip0.333000 \ baselineskip
        

(C)

CHAIN: simple

           \ vskip0.333000 \ baselineskip
        

(D)

TOPOLOGY: linear

           \ vskip0.666000 \ baselineskip
        

(ii)

TYPE OF MOLECULE: DNA (genomic)

           \ vskip0.666000 \ baselineskip
        

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID No. 42:

           \ vskip0.333000 \ baselineskip
        

\dddseqskip

CATAGTGGTC TGCGGAACCG GTGAGT

\hfill

26

 \ dddseqskip 

CATAGTGGTC TGCGGAACCG GTGAGT

 \ hfill 

26

Claims (12)

1. An oligonucleotide to detect acid Hepatitis C virus nucleic wherein said oligonucleotide It has a sequence chosen from the group consisting of

           \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 KY65 \ + SEQ ID NO: 1 \ cr KY98 \ + SEQ ID NO: 3 \ cr KY80 \ + SEQ ID
NO: 5 \ cr KY144 \ + SEQ ID NO: 6 \ cr LY83 \ + SEQ ID NO: 7 \ cr KY84 \ +
SEQ ID NO: 8 \ cr KY85 \ + SEQ ID NO: 9 \ cr KY67 \ + SEQ ID NO: 10 \ cr
KY81 \ + SEQ ID NO: 11 \ cr KY88 \ + SEQ ID NO: 12 \ cr KY86 \ + SEQ ID
NO: 13 \ cr KY87 \ + SEQ ID NO: 14 \ cr KY153 \ + SEQ ID NO: 15 \ cr
KY149 \ + SEQ ID NO: 16 \ cr KY148 \ + SEQ ID NO: 17 \ cr KY78 \ + SEQ ID
NO: 18 \ cr KY145 \ + SEQ ID NO: 19 \ cr KY95 \ + SEQ ID NO: 20 \ cr
KY100 \ + SEQ ID NO: 21 \ cr KY109 \ + SEQ ID NO: 23 \ cr KY111 \ + SEQ
ID NO: 24 \ cr KY99 \ + SEQ ID NO: 25 \ cr KY68 \ + SEQ ID NO: 26, and \ cr
KY82 \ + SEQ ID NO:
27 \ cr}
        

and its complement. 2. The oligonucleotide of claim 1, the which is marked. 3. A method for detecting a hepatitis C virus nucleic acid, characterized in that an oligonucleotide as claimed in claim 1 or 2 is used as a probe. 4. The method of claim 3, which they further comprise an amplification stage, where first amplify a genome sequence of hepatitis virus C, whereby said amplification is preferably obtained with the use of the polymerase chain reaction method. 5. A method for amplifying nucleic acid from a hepatitis C virus, characterized in that an oligonucleotide according to claim 1 or 2 is used as a primer. 6. The method of claim 5, characterized in that a sub sequence of hepatitis C nucleic acid is amplified in the sample using the primer pair:

           \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 KY80 \ + SEQ ID NO: 5 \ cr KY78 \ + SEQ ID NO:
18 \ cr}
        

           \ newpage
        

7. A procedure for detecting a nucleic acid from a hepatitis C virus, characterized in that an oligonucleotide chosen from the group of:

           \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 KY84 \ + SEQ ID NO: 8 \ cr KY85 \ + SEG ID NO: 9 \ cr KY88 \ + SEQ ID
DO NOT:
12 \ cr}
        

it is used as a probe and where a Hepatitis C nucleic acid sequence in the sample is amplify first using the primer pair:

           \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 KY80 \ + SEQ ID NO: 5 \ cr KY78 \ + SEQ ID NO:
18. \ cr}
        

8. A kit to detect the presence of an acid nucleus of a hepatitis C virus, the kit comprising a compartment that contains as an oligonucleotide probe of a oligonucleotide as claimed in claim 1 or 2. 9. A kit to amplify a nucleic acid to from a hepatitis C virus, the kit comprising a compartment containing as primer (s) one or more oligonucleotides claimed in claim 1 or 2, and one or more reagents of reverse transcription or amplification, optionally comprising as probe (s) one or more oligonucleotides according to claim 1 or 2. 10. Use of a compliance oligonucleotide with claim 1 or 2 as a primer. 11. Use of a compliance oligonucleotide with claim 1 or 2 as a probe. 12. Use of a compliance oligonucleotide with claim 1 or 2 to amplify a region 5'-terminal genome of a strain of HCV.